There is considerable interest in understanding the basic immunobiology of T regulatory (Treg) cells as they offer a means to inhibit unwanted immune responses that occur during autoimmune disease or as a consequence of hematopoietic stem cell transplantation (HSCT) or tissue transplant rejection reactions. An important area of Treg cell immunobiology that is poorly understood concerns the antigen specificity of these suppressor T cells in maintaining peripheral self-tolerance. We have developed in vivo model systems that permits direct examination of TCR diversity and specificity as it relates to control of autoimmune disease. In one model, Treg cells are adoptively transferred into IL-2Rb-/- mice, which develop rapid lethal systemic autoimmunity due to their failed production of effective Treg cells. These donor Treg cells fully prevent this autoimmunity and provide a defined population of therapeutic Treg cells to examine issues regarding TCR diversity and specificity. In another clinically relevant model, host Treg cells that survive lethal conditioning after HSCT function to suppress autoreactive donor-derived T cells. We have used these models to address questions concerning the importance of high TCR diversity of Treg cells for self-tolerance. Our initial work shows that in settings of rampant breakdown of immune tolerance, control of autoimmunity is achieved by only a fraction of the available Treg TCR repertoire that was accompanied by substantial peripheral reshaping. Ultimately, too severe constraints on Treg TCR diversity sometimes resulted in autoimmunity. This latter finding is consistent with the hypothesis that TCR repertoire skewing represents a potential intrinsic Treg cell deficit that causes autoimmunity. This proposal plans to build on these data and to exploit the unique features of these models to more precisely define the requirements for Treg TCR diversity and specificity in control of autoimmunity. An important related objective is to investigate mechanisms operative that contribute to peripheral modulation of Treg specificities. To address these issues we propose the following specific aims:1) To further characterize the relevance of TCR diversity in Foxp3+ regulatory T cells by evaluating the efficacy and durability of Treg cells with limited diversity to suppress autoimmunity; 2) to test the requirement for homeostatic mechanisms to drive peripheral reshaping of the Treg TCR repertoire; and 3) to evaluate the effect of altering TCR selection of Treg and autoreactive T cells on peripheral self-tolerance and Treg TCR repertoire reshaping. Completion of these aims should expand our knowledge concerning the selection and diversity of the Treg TCR as it directly relates to the mechanisms by which these cells maintain self-tolerance. This proposal has the potential to impact the field by providing new and essential information that is likely critical for application of Treg cells in immunotherapy to the multitude of scenarios where one wish to inhibit unwanted immune responses. PUBLIC HEALTH RELEVANCE: The utilization of Treg cells in immunotherapy is considered a promising new avenue for highly specific and potentially non-toxic suppression of unwanted immune responses that occur during debilitating autoimmune diseases. Successful therapy with Treg cells requires information concerning the proper TCR specificities that optimally suppress autoreactive T cells. The proposed studies are designed to exploit our novel mouse models to advance understanding concerning the requirement for Treg TCR diversity and specificity in control of autoreactive T cells. The immune system must be continually rebalanced after infections, HSCT, or the use of drugs for immunosuppression and tumor chemotherapy. Indeed, TCR repertoire reshaping occurs for Treg cells after HSCT. Thus, the mechanisms involved in selection and reshaping of the Treg TCR repertoire are fundamental to understand the basis of immune tolerance and susceptibility to autoimmunity and relevant to a widely used clinical therapy. Besides these issues, a rare form of systemic autoimmune disease has been linked to genetic defects in the human IL-2R. Polymorphism in IL-2, IL-2Ra and IL-2Rb are prominent genetic risk factors for a variety of human autoimmune diseases, including multiple sclerosis, type 1 diabetes, rheumatoid arthritis and celiac disease. The IL-2Rb-deficient model used in some of these studies also has an important link to human autoimmunity. Understanding the factors that reverse autoimmunity in the IL-2Rb- deficient model may also provide new information concerning the etiology and potential therapy that may be generally common to several human autoimmune diseases.